CHOLERA--PATHOGENESIS AND IMMUNOLOGY

霍乱——发病机制和免疫学

• 批准号: 2060489
• 负责人: Richard A. Finkelstein
• 金额: $ 25.65万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2060489
• 关键词: Vibrio cholerae; bactericidal immunity; chickens; chimeric proteins; cholera; cholera toxin; cholera vaccine; endopeptidases; host organism interaction; laboratory mouse; laboratory rabbit; lipopolysaccharides; microorganism growth; microorganism hemagglutinin; protein engineering; protein sequence; site directed mutagenesis; synthetic protein; virulence

The goals of this research are to contribute to the understanding of the pathogenesis of cholera, an epidemic diarrheal disease caused by Vibrio cholerae 01, at both the organismal and the molecular levels, with the expectation that this information will lead to the development of rational and effective means of immunoprophylaxis. As cholera is the prototype of an expanding number of diarrheal diseases which are mediated by enterotoxins which are structurally, functionally and immunologically related to the cholera enterotoxin(s), the observations are pertinent to the larger global problem of the billions of cases and millions of deaths annually from diarrheal diseases especially in children in the Third World. Cholera enterotoxin (CT) was discovered in 1959 and it was purified and partially characterized in 1969. In the ensuing period, expectations that, as with diptheria and tetanus, a toxoid vaccine would prevent the disease have not been fulfilled despite early encouraging experimental observations. It is now known that the disease, cholera, is an effective immunizing process in which the human host is presented with a consortium of products elaborated by the cholera vibrios growing in vivo in/on the small bowel. In addition to the enterotoxin, these include colonization factors, such as toxin-coregulated pili (TCP), other surface components including lipopolysaccharide (LPS) and outer membrane proteins, HA/protease and other soluble factors, the cell-associated mannose-sensitive hemagglutinin of the E1 Tor biotype, the flagellum and, possibly, other factors. The solid immunity evoked in people by the disease has not yet been duplicated by non-living preparations, administered parenterally or perorally, or by living attenuated mutants, administered perorally, although some degree of protection has been attained -- even in the absence of toxin antigen. Results of studies with toxin antigen by itself have been even more disappointing. This proposal addresses some of the reasons for previous failures of toxoid vaccines; it addresses the development of a new cholera vaccine to consist of a non-toxic conjugate of de-lipidated LPS and cholera toxin; and, in addition, it will continue to address other factors which may contribute to the virulence and immunogenicity of wild- type cholera vibrios in vivo. The technology includes the synthesis and analysis of sequential continuous overlapping peptides comprising the immunologically dominant B-subunit protein of CT and the use of site- specific mutagenesis to create a V. cholerae strain which produces a CT which is not toxic but expresses the major epitopes of the holotoxin. This CT will also be cross-linked to LPS oligosaccharide to form a conjugate vaccine which will stimulate both antibacterial and antitoxic immunity. We will further examine and define the role of HA/protease and the mannose- sensitive hemagglutinin in attachment and/or detachment of cholera vibrios and examine the significance of phase variation in opacity of V. cholerae colonies.

这项研究的目标是为理解 霍乱的发病机理，霍乱是一种流行性腹泻疾病 霍乱01，在生物和分子水平上，与 期望这些信息将导致理性发展 和有效的免疫保障手段。 因为霍乱是 扩大数量的腹泻疾病由 在结构，功能和免疫学上都是肠毒素 与霍乱肠毒素有关，观察结果与 数十亿起病例和数百万死亡的全球问题更大 尤其是在第三世界的儿童中，每年都患有腹泻疾病。 霍乱肠毒素（CT）于1959年发现，并被纯化并且 部分在1969年进行了特征。在随后的时期，期望 与白喉和破伤风一样，毒素疫苗会预防这种疾病 尽管早期鼓励实验，但尚未实现 观察。 现在众所周知，霍乱是一种有效的 呈现人类主机的免疫过程 在体内生长在/上的霍乱弧菌所详细阐述的产品 小肠。 除肠毒素外，这些还包括定植 因素，例如毒素调节的pili（TCP），其他表面成分 包括脂多糖（LPS）和外膜蛋白，HA/蛋白酶 和其他可溶性因素，与细胞相关的甘露糖敏感 E1 tor Biotype，鞭毛和其他可能的Hemagglutinin 因素。 疾病中尚未引起人们的固体免疫力 通过非生物制剂重复，属于育儿或 经地或通过活着的衰减突变体，经常给予 尽管已经获得了某种程度的保护 - 即使在不在的情况下 毒素抗原。 毒素抗原的研究结果本身具有 更令人失望。 该提议解决了一些原因 对于先前的毒素疫苗失败；它解决了一个发展 新的霍乱疫苗，由脱脂lps的无毒结合物组成 和霍乱毒素；而且，此外，它将继续解决其他 可能导致野生毒力和免疫原性的因素 在体内键入霍乱弧菌。 该技术包括合成和 分析包含该肽的连续重叠肽 CT的免疫学显性b-subunit蛋白以及使用部位的使用 特定的诱变，以产生霍乱链球菌，产生CT 这是无毒的，但表达了Holotoxin的主要表位。 这 CT还将交联至LPS寡糖以形成共轭 疫苗会刺激抗菌和抗毒性免疫。 我们 将进一步检查并定义HA/蛋白酶和甘露糖的作用 敏感的血凝集素在附着和/或脱离霍乱弧菌中 并检查相位变化对霍乱弧菌不透明度的重要性 殖民地。